WO2011051322A1 - Cloning, expression and use of acid phospholipases - Google Patents

Abstract

The invention relates to a DNA sequence, which codes for a polypeptide having phospholipase activity essentially without lipase activity, characterized in that the DNA sequence is selected from a) DNA sequences that comprise a nucleotide sequence according to SEQ ID No: 1, b) DNA sequences that comprise the coding sequence according to SEQ ID NO: 1, c) DNA sequences that code for the protein sequence according to SEQ ID NO: 2, d) DNA sequences that are coded for with the restriction map by the pPL3940-Topo2.5 plasmid according to figure 7 and that are stored under number DSM 22741, e) DNA sequences that hybridize under stringent conditions with one of the DNA sequences according to a), b), c) or d), f) DNA sequences that are related to the DNA sequences according to a), b), c), d) or e) in the basis of the degeneration of the genetic code, and g) complementary strands to the sequences according to a) to f), wherein the DNA sequence is preferably derived from Aspergillus, and more preferably from Aspergillus fumigatus, and a polypeptide having phospholipase activity essentially without lipase activity selected from a) a polypeptide which is coded for by the coding part of a DNA sequence as defined above, b) a polypeptide having the sequence according to SEQ ID NO: a or a sequence derived therefrom, which can be obtained by substituting, adding, or deleting one or more amino acids, c) a polypeptide having a sequence that has at least 83% identity with the amino acids 1 to 299 of SEQ ID NO: 2, d) a polypeptide for which a nucleic acid sequence codes, which is hybridized under stringent conditions with (i) nucleotides 55 to 1106 of SEQ ID NO: 1, (ii) the cDNA sequence contained in nucleotides 55 to 1106 of SEQ ID NO: 1, (iii) a partial sequence of (i) or (ii) composed of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii) or (iii), e) a variant of the polypeptide having SEQ ID NO: 2, comprising a substitution, deletion and/or insertion of one or more amino acids, f) allelic variants for amino acid sequences a) to e).

Description

 Cloning, expression and use of acid phospholipases The invention relates to novel DNA sequences which encode polypeptides having phospholipase activity substantially without lipase activity. The invention further relates to novel polypeptides having phospholipase activity substantially without lipase activity. These polypeptides are low molecular weight acid phospholipases with high thermostability and high temperature stability, respectively. These polypeptides are also active over a wide pH range. Furthermore, the invention also relates to the use of these phospholipases for the reduction of phosphorus-containing compounds, for example in the production of edible oils and the use of these phospholipases as baking aids, animal feed auxiliaries, auxiliaries in textile raw material processing, etc.

Raw vegetable oils contain impurities (e.g., free fatty acids, phospholipids, heavy metals, dyes, ...) which, when stored by the hydrolytic and oxidative alteration of lipids, compromise the quality and durability of the oil and make further processing difficult. Therefore, a refining after the extraction of crude vegetable oils to eliminate unwanted impurities is necessary. In the production of high quality edible oils, refining involves the steps of degumming, bleaching and deodorization. The first step in the refining of oils is degumming. The degumming is used to remove mucilage, primarily phospholipids, which cause a negative change in the taste of the oil and interfere with the further process steps of the oil refining. Since the phosphorus content is a measure of the degree of degumming, the quality of the degummed oil obtained can be assessed by determining the residual phosphorus content. A degummed oil containing less than 10ppm residual phosphorus is required in further refining operations.

Phospholipids are complex, phosphorus-containing lipids. Phospholipids, such as phosphatidylcholines or lecithin, consist of a glycerol backbone esterified with fatty acids in positions sn-1 and sn-2 and with an ester-linked phosphate group in position sn-3. The phosphate group in turn can again, for example, be esterified with a primary alcohol group. Natural phospholipids contain at the positions sn-1 and sn-2 different fatty acid chains, which are predominantly the polyunsaturated acyl chains in the plant material. There are two types of phospholipids, hydratable and non-hydratable. For the removal of phospholipids different methods are used.

The simplest method is water degumming. In this method, hydratable phospholipids can be washed out with water and removed from the oil. Depending on the type and quality of the crude oil, the degummed oil still contains 80-200 ppm phosphorus after this process.

In the acid degumming method, the oil is treated with acid. The acid causes non-hydratable phospholipids to be converted to hydratable phospholipids. The hydratable phospholipids become oil insoluble. It forms oil insoluble sludge, which is removed from the oil by centrifugation or filtration. According to this method, the residual phosphorus content in the degummed oil is about 25-100 ppm. Enzymatic degumming provides an efficient, inexpensive and environmentally friendly process for gently removing the phospholipids from edible oil.

Phospholipases are enzymes which cleave phospholipids and are divided according to their enzymatic cleavage sites on phospholipid into acyl hydrolases (phospholipase A1, A2 and B) and phosphodiesterases (phospholipase C and D). Phospholipase A1 (EC 3.1 .1 .32) hydrolyzes the fatty acid at the sn-1 position of phospholipid molecules, and phospholipase A2 (EC 3.1 .1 .4) specifically cleaves the sn-2 ester bond from the phospholipid. The reaction products are the lysophospholipid and the free fatty acid. Phospholipase B (EC 3.1 .1 .5) nonspecifically cleaves the fatty acid at both sn-1 and sn-2 positions. Phospholipase C (EC 3.1 .4.10) hydrolyzes the phosphate ester bond between glycerol and the phosphate group to phosphate monoester and diacylglycerol. Phospholipase D (EC 3.1 .4.4) catalyzes the hydrolysis of the terminal phosphodiester bond to give phosphatidic acid and choline as cleavage products.

It is known that the production of phospholipase A2 from bovine and porcine pancreas, from the venom of the honeybee or various types of snakes. Phospholipase may also be derived from microorganisms, eg from bacteria and fungi and produced by recombinant techniques with sufficient yield.

In the patent EP 0 513 709 an effective enzymatic degumming process is presented for the first time. The new method uses for the first time the enzyme phospholipase A2, which cleaves the fatty acid at the sn-2 position of phospholipids. The procedure was tested on soy and rapeseed oil with a phosphorus content of 72 to 1 10 ppm. The reaction contained up to 5% by weight (w / w) of water, based on the oil, and was incubated at pH 5.0-5.5 at 40 ° C and 60 ° C, respectively, for up to 5 hours. The resulting lysophospholipid can be removed by centrifugation from the oil. The degummed oil then has a residual phosphorus content of less than 5 ppm.

During degumming, a defined amount of water is added to the oil. Then the enzyme-containing water phase and the oil phase are mixed together, so that the enzyme can act. The amount of water should be kept as small as possible. The use of large amounts of water means increased energy consumption and increased disposal costs. Therefore, a process of enzymatic degumming with low water content (2%) is already advantageous in this respect.

US2007 / 134777 states that the enzymatic degumming of vegetable oil with phospholipase A1 is carried out at a pH between 4.0 and 5.0. The optimum pH for this reaction is between 4.5 and 5.0. In this pH range, the liberated calcium and / or magnesium ions can combine with other chemicals (anions) of the reaction buffer to poorly soluble salts, which are deposited on the surface of the reactors and thereby pollute the device. The elimination of such contamination and the cleaning of the device are expensive. In order to reduce contamination of the equipment interior, the reaction is preferably carried out at a pH of about 4. However, the enzyme becomes less active or functionally inactive as the pH of the reaction continues to decrease.

EP 0 904 357 describes that a phospholipase A was found in Aspergillus niger for degumming edible oil.

In WO2008 / 040466 it is described that in Aspergillus fumigatus a phospholipase with a molecular weight of 65 kDa for degumming Edible oil with 5% water content up to temperatures of 65 C can be used.

EP 1 788 080 describes the use of phospholipase C from Bacillus cereus in the degumming of oil in 6 hours at 60 ° C with a water content of 15% based on the oil. The residual phosphorus content is then less than 5 ppm.

In WO2008 / 094847 it is described that the reaction time of the oil degumming under the interaction of the phospholipase A1 or A2 with the phospholipase C can be shortened to 30 min.

Phospholipases are also widely used in the food and feed industries, e.g. in the dough production, in the production of baked goods, to increase the yield in the cheese production, etc. Therefore, phospholipases are needed, which can be used technologically versatile.

In biotechnology, phospholipases are used as biocatalysts for the production of phospholipids. Phospholipids are polar lipids and act as emulsifiers due to lipophilic and hydrophilic structural features. An example is the use of phospholipases in the production of modified lecithins, as food emulsions in the production of sauces, mayonnaise and salad dressing, in the production of instant powders, for example milk, cocoa and coffee powders, as flow improvers in chocolate production and as dietary supplements. In the pharmaceutical and cosmetic industries, the phospholipids and lysophospholipids are used in the preparation of creams, lotions, gels and liposome formulations.

Lecithin is also needed to make lacquers, paints, magnetic tapes, specialty papers, leather and textiles. Phospholipases are also used in the textile industry in the "Bioscouring" for the purification of the plant fiber before further processing steps such as coloring.Also, a mixture of phospholipase can be used together with other enzymes.The other enzymes can be from the group of cellulases, hemicellulases , Pectinases, proteases, and oxidoreductases. There is therefore a constant need in the art for phospholipases with the widest possible or optimally optimized field of use.

The object of the present invention is therefore to provide proteins or polypeptides having improved phospholipase properties. In particular, the novel phospholipases should not have any lipase activity relevant in technological processes. In particular, the proteins with phospholipase activity should be active over a wide pH range or have a high temperature resistance.

Furthermore, the proteins with phospholipase activity should be simple, inexpensive and economical to produce. Furthermore, according to the invention expression constructs are to be provided which are suitable for the production of proteins with phospholipase activity.

The above objects are achieved by a DNA sequence which encodes a polypeptide having phospholipase activity substantially without lipase activity, characterized in that the DNA sequence is selected from a) DNA sequences which comprise a nucleotide sequence according to SEQ ID NO: 1, b) DNA sequences comprising the coding sequence of SEQ ID NO: 1, c) DNA sequences encoding the protein sequence of SEQ ID NO: 2, d) DNA sequences derived from the plasmid pPL3949-Topo2.5 with the restriction map according to FIG. 7 and deposited under the accession number DSM 22741, e) DNA sequences which hybridize under stringent conditions with one of the DNA sequences according to a), b), c) or d), f) DNA sequences Sequences related to the DNA sequences according to a), b), c), d) or e) due to the degeneracy of the genetic code, and g) complementary strands to the sequences according to a) to f). The invention further relates to a polypeptide with phospholipase activity substantially without lipase activity, selected from a) a polypeptide which is encoded by the coding part of one of the above DNA sequences, b) a polypeptide with the sequence according to SEQ ID NO: 2 or one of them c) a polypeptide having a sequence which has at least 83% identity to the amino acids 1 to 299 of SEQ ID NO: 2, d) a polypeptide, c) a polypeptide which is obtainable by substitution, addition or deletion of one or more amino acids thereof; which is encoded by a nucleic acid sequence which hybridizes under stringent conditions to (i) nucleotides 55 to 1106 of SEQ ID NO: 1, (ii) of the cDNA sequences contained in nucleotides 55 to 1106 of SEQ ID NO: 1 Sequence, (iii) a partial sequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii) or (iii), e) a variant of the polypeptide of SEQ ID NO: 2 comprising a substitution, deletion and / or insertion of one or more amino acids, f) allelic variants to the amino acid sequences a) to e).

Furthermore, the invention relates to expression constructs or hosts which are capable of expressing the polypeptides according to the invention having phospholipase activity. Furthermore, the invention also relates to the corresponding expression plasmids and vectors. Furthermore, the invention relates to methods for degumming vegetable oil using the polypeptides of the invention and the use of the polypeptides according to the invention for applications in the field of food technology, in particular for the production of dough, baked goods or dairy products, or in animal nutrition and in the processing of textile raw materials, the so-called Scouring or Bioscouring.

According to a further embodiment, the invention relates to a polypeptide with phospholipase activity substantially without lipase activity, which is characterized in that it has a molecular weight in the range of 28 to 30 kDa and preferably of about 28.6 kDa, a broad pH optimum and a has high temperature resistance and is isolatable from an organism of the genus Aspergillus.

It is to be understood by high temperature resistance that the enzyme after 6 h at a temperature of 60 ° C under the conditions at the Ölentschleimung with a low water content of 2% retains its activity in an industrially utilizable extent. The enzyme retains its industrially utilizable activity by resulting in oils with technologically virtually insignificant residual phosphorus contents. In this case, the residual phosphorus content in the enzymatically degummed oil is preferably less than 10 ppm, more preferably less than 5 ppm.

Surprisingly, it has been found that a DNA sequence encoding a polypeptide having phospholipase activity substantially without lipase activity, which has a low molecular weight and a high temperature resistance, can be isolated from a strain of the genus Aspergillus fumigatus. This phospholipase is an acidic filamentous fungal phospholipase having a calculated molecular weight of about 28.6 kDa capable of hydrolyzing at least one of the two fatty acids from lecithin. In contrast to the polypeptides with phospholipase activity, which are known from the prior art, the phospholipases according to the invention have a high temperature resistance (at 60 ° C) and can thereby advantageously also in processes of enzymatic degumming with a low water content of 2% (based on Oil) and at a pH of 4.0. This is of particular economic interest because, therefore, in the degumming processes, the temperature of the oil need not first be lowered to allow enzymatic degumming without inactivation of the enzyme, and then the temperature of the oil must be increased to increase the viscosity of the oil for the centrifugation step to reduce the separation of the oil and water phases. The temperature stability of the polypeptides according to the invention having phospholipase activity is also advantageous for other applications in the field of food technology and animal nutrition or in textile processing.

Prior art phospholipases are excluded from the scope of the invention. It is therefore very advantageous that the A. fumigatus-derived phospholipases according to the invention are active in a wide pH range of 3 to 5 or show a very broad optimum of activity in this range. The phospholipases according to the invention thus not only have the advantage that they have essentially no lipase activity. They also develop their enzyme activity over a wide pH range and can thus be used over a wide pH range.

Enzymes with phospholipase activity (phospholipase A, B, C, or D) from Aspergillus fumigatus have been reported to date in various publications (Birch et al., Comparison of extracellular phospholipase activities in dinical and environmental Aspergillus fumigatus isolates, 2004, Med Mycol 42 (1): 81-86; Rementeria et al., Genes and molecules involved in Aspergillus fumigatus virulence, 2005, Rev. Iberoam Micol 22 (1): 1-23), but not accurately characterized. From the Aspergillus fumigatus genome sequence (Nierman et al., Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus, 2005, Nature, 438 (7071): 151-6), several hypothetical phospholipase- For example, a hypothetical phospholipase A with 241 amino acids and three high molecular weight lysophospholipases Plb1, Plb2, and Plb3 was shown, and the database also shows a small hypothetical extracellular lipase (A, B, C, D) and lysophospholipase genes 299 amino acids and a calculated molecular weight of about 28.5 kDa (GenBank EAL86100) and several lipases, which have between 409 to 587 amino acids. Shen et al. (Characterization and expression of phospholipase B from the opportunistic fungus Aspergillus fumigatus, 2004, FEMS Microbol Lett 239 (1): 87-93), succeeded in the cloning and characterization of 3 phospholipase B genes from Aspergillus fumigatus. The secreted proteins AfPL1 with 633 amino acids and AfPL3 with 630 amino acids have a molecular weight of about 68 kDa. The protein AfPL2 is a cytosolic protein with 588 amino acids and has a molecular weight of about 63 kDa.

From Aspergillus fumigatus RH3949 were further isolated a phosphopholipase with 633 amino acids (WO2008 / 040466) and a lysophospholipase with 61 1 amino acids (WO2008 / 040465).

Furthermore, a small molecular weight acid phospholipase (pI 4.1) in the range of 28 to 30 kDa was found in the genome of Aspergillus fumigatus RH3949. In the protein sequencing of this "small" phospholipase, the first 16 amino acids at the N-terminus showed a 93% identity to the N-terminal sequence of the hypothetical 299 amino acid extracellular lipase (GenBank EAL86100) from Aspergillus fumigatus Af293. 299) The sequence of the "small phospholipase" from RH3949 with the sequence of the hypothetical extracellular lipase is only about 82%. This was particularly surprising, since at a comparable location in the genome of another Aspergillus fumigatus strain (A1 163) a sequence with considerably higher identity to the hypothetical lipase from Af293 was found (Genbank EDP1054) (Fedorova et al., Genomic Islands in the Pathogenic Filamentous Fungus Aspergillus fumigatus, 2008, PloS Genet.4, e1000046).

In contrast, there is no match between two N-terminal phospholipase sequences from Aspergillus fumigatus Af293 (241 amino acid phospholipase A, GenBank EAL85761) and Aspergillus fumigatus RH3949 (phospholipase with pI 4.1).

Thus, the phospholipase according to the invention differs from both known phospholipases from Aspergillus fumigatus as well as lipases annotated sequences of very closely related Aspergillus fumigatus strains such as Af293.

From the DNA sequence data (GenBank AAHF0100001 1) for the hypothetical extracellular lipase (GenBank EAL86100) various oligonucleotide primers were inventively derived and synthesized.

Surprisingly, it was found that with primers derived from the genome sequence of the strain Af293, the DNA sequence according to the invention from the Aspergillus Stannnn RH3949 could be isolated (see Example 4). This was not expected because the strain RH3949 (an environmental isolate) of Af293 (clinical isolate) used for amplification differs phenotypically and thus most likely genotypically (sequence). Thus it was not possible to amplify the phospholipase gene from RH3949 with other primer pairs with binding sites on N2-3948 and Apal-3949 on the genomic DNA of strain Af293.

The amplification of the gene was carried out by means of the polymerase chain reaction (PCR) from genomic DNA of Aspergillus fumigatus RH3949.

The temperature resistance and the broad pH optimum of the phospholipase according to the invention were surprising and could not be expected on account of the phospholipases described in the prior art. For none of the naturally occurring phospholipases of filamentous fungi described in the prior art these properties are described or even suggested.

The phospholipase sequence of SEQ ID NO: 2 according to the invention was compared with phospholipase sequences of the prior art. A partial match at the amino acid sequence level was found to known amino acid sequences from other / 4sperg / V / us strains, i. the agreement was 60% with a lipase from Aspergillus tubingensis (WO98 / 45453) or with a lysophospholipase from Aspergillus foetidus (EP0808903), 59% with an Aspergillus niger phospholipase (WO03 / 097825, WO98 / 31790).

The sequence SEQ ID NO: 2 shows the highest identity of 82% with a hypothetical extracellular lipase sequence (Genbank EAL86100) from Aspergillus fumigatus Af293 (Nierman et al., Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus, 2005, Nature, 438 (7071): 1 151-6).

Surprisingly, the phospholipase isolated from Aspergillus fumigatus RH3949 according to the invention has no lipase activity relevant for this process under the conditions of enzymatic degumming of edible oil. Furthermore, in contrast to previously known phospholipases from other Aspergillus species, this enzyme has an exceptionally broad pH optimum and a high temperature resistance. The enzyme according to the invention can thus be used advantageously in a process for the enzymatic degumming of edible oils, since it hydrolyzes no or only insignificant proportions of triglyceride bonds in the oil.

The invention further relates to polypeptides having phospholipase activity substantially without lipase activity with a sequence having at least 83% identity to the sequence according to SEQ ID NO: 2. Preferably, the invention relates to a polypeptide having phospholipase activity with a sequence having at least 83% identity to amino acids 1 to 299 of SEQ ID NO: 2. Preferably, the degree of identity to amino acids 1 to 299 of SEQ ID NO: 2 is at least 90%, more preferably at least 95%, even more preferably at least 97%, and most preferably at least 98%, provided that the respective sequences have phospholipase activity substantially without lipase activity exhibit.

The polypeptides according to the invention having phospholipase activity have no appreciable lipase activity or are essentially without lipase activity. The polypeptides according to the invention essentially have no lipase activity which is detrimental to industrial processes of oil degumming, ie the polypeptides according to the invention exhibit substantially no activity against lipolytically cleavable compounds in the oil to be degummed. This means that under the conditions of the enzymatic degumming of edible oil, the phospholipases according to the invention have no lipase activity relevant for this process. Technologically, this means that the polypeptides according to the invention having phospholipase activity hydrolyze p-nitrophenyl palmitate as lipase substrate only to an insignificant and / or undetectable extent. For the polypeptides of the invention having phospholipase activity, the ratio of phospholipase activity to lipase activity is preferably> 1, 000: 1, more preferably 5,000: 1 to 10,000: 1, even more preferably 7,000: 1, most preferably 7,500: 1. The degree of sequence identity is preferably determined by determining the number of residues of the shorter sequence involved in the comparison and having a "corresponding" counterpart in the other sequence For the purposes of the present invention, the identity will be included In accordance with the invention, only the cDNAs or amino acids of the respective mature proteins are used for comparison In homologous sequences similar, preferably identical, sequence counterparts were determined according to the invention with the aid of known computer programs Program is the program Clone Manager Suite, which contains the program part Align Plus and is distributed by Scientific & Educational Software, Durham, NC, USA, comparing two DNA or amino acid sequences as defined above, under the option local alignment either n After the method FastScan - MaxScore or after the method Needleman-Wunsch while maintaining the default values. Specifically, the program version "Clone Manager 7 Align Plus 5" with the functions "Compare Two Sequences / Local Fast Scanning Max Score / Compare DNA sequences", or for amino acids "Compare Two Sequences / Global / Compare sequences As Amino Acids "the algorithms available from the following sources were used: Hirschberg, DS 1975. A linear space algorithm for computing maximum common subsequences Commun Assoc Comput Mach 18: 341-343, Myers, EW and W. Miller. 1988. Optimal alignments in linear space, CABIOS 4: 1, 11-17, Chao, KM, WR Pearson and W. Miller, 1992. Aligning two sequences within a specified diagonal band, CABIOS 8: 5, 481-487.

The invention further relates to addition and / or deletion molecules of the above polypeptides having phospholipase activity. Thus, a polypeptide modified according to the invention having phospholipase activity can be extended by adding further sequences at the N-terminal and / or C-terminal end, wherein the amino acid sequences thus obtained must still have phospholipase activity substantially without lipase activity. As a result, hybrid molecules can be produced which have further advantageous properties. For example, suspension proteins or their native precursor forms can be added to highly secreted proteins, thereby further improving secretion efficiency. Further, active sequence portions of other enzymes can be added to produce multi-specificity enzymes. Furthermore, polar or non-polar sequences can be added in order to influence the solubility properties or the membrane permeability of the enzyme thus obtained in a targeted manner. Sequence sections of the polypeptide with phospholipase activity can also be deleted according to the invention while maintaining the phospholipase activity essentially without lipase activity. The mutations, elongations and truncations can be carried out in a manner known per se according to methods well known in the art. Truncated polypeptides are often characterized by an improved secretion level compared to the full-length polypeptides. They may also have higher thermal stabilities compared to the full-length polypeptide because they contain only the "compressed core." The generation of such variants is well known in the art For example, amino acid sequence variants of the polypeptides can be made by mutation in the DNA and nucleotide sequence alteration are well known in the art (see, for example, Tomic et al., NAR, 18: 1656 (1990), Giebel and Sprtiz NAR, 18: 4947 (1990)).

Evidence of suitable amino acid substitutions that do not interfere with the biological activity of the protein of interest can be found in the model of Dayhoff et al., Atlas of Protein Sequence and Structure, Natl. Biomed. Res. Found., Washington, D.C. (1978). Conservative substitutions such as the replacement of one amino acid for another with similar properties are preferred. These exchanges can be divided into two main groups with a total of four subgroups, and an exchange in each subgroup is referred to as conservative exchange, which preferably does not affect the activity or folding of the protein.

The terms "protein,""peptide," and "polypeptide" are used essentially interchangeably A polypeptide or enzyme with phospholipase activity or a phospholipase is intended to mean an enzyme that catalyzes the release of fatty acids from phospholipids, for example, lecithins can be determined by using any of the known measuring methods using one of these substrates.

In connection with the polypeptides according to the invention, the term "phospholipase" or phospholipase A is intended to denote both enzymes having phospholipase A1 and phospholipase A2 activity, phospholipase A1 or A2 being defined according to the standard enzyme EC classification as EC 3.1.1 .32 or 3.1 .1 .4.

Phospholipase B or lysophospholipase are polypeptides according to standard enzyme EC classification EC 3.1 .1 .5.

The invention further relates to DNA sequences encoding a polypeptide having phospholipase activity comprising mutations, modifications or variations of the sequence according to SEQ ID NO: 1. Furthermore, the invention also relates to sequences which hybridize under relaxed or stringent conditions with the above sequences. Stringent conditions are: hybridization at 65 ° C., 18 h in dextran sulfate solution (GenescreenPlus, DuPont), then the filter is washed for 30 minutes first with 6 × SSC, twice 2 × SSC, twice 2 × SSC, 0.1% SDS and then with 0, 2 x SSC at 65 ° C (membrane transfer and detection methods, Amersham).

Furthermore, the invention also relates to DNA sequences which are related due to the degeneracy of the genetic code with the above sequences of the invention, and allelic variants thereof. The degeneracy of the genetic code may be due to natural degeneration or due to a specially chosen codon usage. Naturally occurring allelic variants can be identified using well known techniques of molecular biology, such as polymerase chain reaction (PCR) and hybridization techniques.

The invention further relates to a method of producing a polypeptide having phospholipase activity by recombinant techniques comprising culturing recombinant prokaryotic and / or eukaryotic host cells containing a DNA sequence of the invention under conditions which promote expression of the enzyme, and then recovering the enzyme. The invention further relates to the use of the polynucleotide sequences of the invention for the production of probes for finding similar sequences encoding corresponding enzymes in other organisms as well as for the transformation of host cells. A DNA sequence encoding a polypeptide of the invention can be used to transform any host cells, such as fungi, yeasts, bacteria, plants, or mammalian cells. Such transformed cells are characterized by a secretion of the phospholipase according to the invention. The phospholipase enzyme thus produced causes an efficient hydrolysis of the fatty acids from phospholipids.

The invention also relates to expression cassettes which can be used to introduce a phospholipase encoding a DNA sequence of the invention or an open reading frame into a host cell. They preferably comprise a transcription initiation region linked to the open reading frame. Such an expression cassette may have a plurality of restriction sites for insertion of the open reading frame and / or other DNAs, e.g. a transcriptional regulatory region and / or selectable marker genes. The transcriptional cassette comprises in the 5 'to 3' direction of transcription a transcriptional and translational initiation region, the DNA sequence of interest, and a transcriptional and translational stop region that is functional in a microbial cell. The termination region may be native to the transcription initiation region, may be native to the DNA sequence of interest, or may be derived from any other source.

The term "open reading frame" (ORF) refers to the amino acid sequence encoded between the translation start and stop codons of a coding sequence The terms "start codon" and "stop codon" designate a unit of three contiguous ones Nucleotides (codons) in a coding sequence that specify the chain start and stop of protein synthesis (mRNA translation).

"Functional linkage" in the context of a nucleic acid refers to a compound as part of the same nucleic acid molecule in proper position and orientation for the transcriptional initiation of the promoter DNA operably linked to a promoter is under the transcriptional initiation regulation of the promoter With respect to polypeptides, functional linkage means the compound as part of the same polypeptide, ie via peptidyl bonds.

Any promoters can be used according to the invention. Promoter usually refers to the nucleotide sequence upstream (5 ') with respect to coding sequence and controls expression of the coding sequence by providing recognition for the RNA polymerase and other factors required for proper transcription. The promoter used according to the invention may comprise a minimal promoter, ie a short DNA sequence from a TATA box and other sequences specifying the transcription start site, to which regulator elements for expression control are attached.

The promoter used in the invention may also comprise a nucleotide sequence comprising a minimal promoter and regulatory elements capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and distal upstream elements, the latter elements often being referred to as enhancers. Thus, an enhancer is a DNA sequence that can stimulate promoter activity and may be an element inherent to the promoter or an inserted heterologous element to enhance the level of expression or tissue specificity of a promoter. It can function in both orientations and can function even on upstream or downstream placement of the promoter. Both enhancers and other upstream promoter elements sequence-specifically bind DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or may be composed of various elements derived from various naturally occurring promoters, or may even be composed of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the efficiency of transcription initiation in response to physiological or developmental conditions. Promoter elements, especially TATA elements, which are inactive or have greatly reduced promoter activity in the absence of upstream activation are termed minimal promoters or core promoters. In the presence of a suitable transcription factor or transcription factors, the function of the minimal promoter is to allow transcription. A minimal or core promoter thus consists only of all basic elements necessary for transcription initiation, e.g. As a TATA box and / or an initiator. The invention also relates to vector sequences containing DNA sequences according to the invention. These vector constructs include any plasmids, cosmids, phage and other vectors in double-stranded or single-stranded, linear or circular form which may optionally be self-transmissive or mobilizable and which may transform a prokaryotic or eukaryotic host either by integration into the cellular genome or which are extra-chromosomally present (eg, autonomously replicating plasmids with a replication origin). Vectors, plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and DNA segments for use in transforming cells generally comprise the DNA encoding the phospholipase of the present invention, as well as other DNA such as cDNA, a gene, or genes found in the cells are to be introduced. These DNA constructs may include other structures such as promoters, enhancers, polylinkers or regulatory genes, as necessary. One of the DNA segments or genes selected for cellular delivery will usefully encode / encode a protein expressed in the transformed (recombinant) cells thus obtained, resulting in a screenable or selectable trait and / or conferring an improved phenotype to the transformed cell.

The construction of vectors which can be used in accordance with the invention is known to one skilled in the art in light of the above disclosure and general knowledge (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed. Cold Spring Harbor Laboratory Press, Plainview, NY (1989)).

An expression cassette according to the invention may contain one or more restriction sites in order to place the phospholipase-encoding polynucleotide under the regulation of a regulatory sequence. The expression cassette may also contain a termination signal operably linked to the polynucleotide as well as regulatory sequences needed for proper translation of the polynucleotide. The expression cassette containing the polynucleotide according to the invention may be chimeric, ie at least one of its components is heterologous with respect to at least one of the other components. The expression of the polynucleotide in the expression cassette may be under the control of a constitutive promoter, an inducible promoter, a regulated promoter, a viral promoter or a synthetic promoter.

The vectors may already contain regulatory elements, e.g. Promoters, or the DNA sequences of the invention, can be manipulated to contain such elements. Suitable promoter elements which can be used are known in the art and are for example Trichoderma reesei the cbhl or the cbh2 promoter, for Aspergillus oryzae the amy promoter, for Aspergillus niger the xyl, glaA, alcA, aphA , tpiA, gpdA, sucl and the pkiA promoter. Suitable promoter elements that can be used for expression in yeast are known in the art and are, for example, the pho5 promoter or the gap promoter for expression in Saccharomyces cerevisiae and for Pichia pastoris, e.g. the aoxI promoter or the fmd promoter or the mox promoter for H. polymorpha.

DNA suitable for introduction into cells, in addition to the DNA of the invention, may also comprise DNA derived from or isolated from any source. An example of a deduced DNA is a DNA sequence which has been identified as a useful fragment in a given organism and which has then been chemically synthesized in substantially pure form. An example of such a DNA is a suitable DNA sequence obtained, for example, using restriction endonucleases so that it can be further manipulated according to the invention, for example amplified. These include, among others, the amdS gene from Aspergillus nidulans, which can be used as a marker gene, and its regulatory sequences, as well as polylinkers.

Such DNA is commonly referred to as recombinant DNA. Thus, a suitable DNA comprises wholly synthetic DNA, semisynthetic DNA, DNA isolated from biological sources, and DNA derived from in-blood RNA. In general, the introduced DNA is not an original part of the genotype of the recipient DNA, but according to the invention a gene from a given genotype can also be isolated and eventually altered and subsequently multiple copies of the gene can be introduced into the same genotype, e.g. To enhance production of a given gene product.

The introduced DNA includes, without limitation, DNA from genes such as bacteria, yeasts, fungi or viruses. The introduced DNA can be modified or synthetic genes, parts of genes or chimeric genes including genes from the same or a different genotype. For this purpose, z. As well as DNA of the plasmids pUC18, pUC19 belong.

The DNA used for the transformation according to the invention may be circular or linear, double-stranded or single-stranded. In general, the DNA is in the form of a chimeric DNA, such as plasmid DNA, which also contains coding regions flanked by regulatory sequences which aid in the expression of the recombinant DNA present in the transformed cell. For example, the DNA itself may contain or consist of a promoter that is active in a cell derived from a source other than the cell, or a promoter that is already in the cell, i. the transformation target cell is present.

In general, the introduced DNA is relatively small, less than about 30 kb, to minimize sensitivity to physical, chemical or enzymatic degradation, which increases with the size of the DNA.

The selection of a suitable expression vector depends on the host cells. Yeast or fungal expression vectors may include a replication origin, a suitable promoter and enhancer, and also include any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and non-transcribed 5 'flanking sequences. Examples of suitable host cells are: fungal cells of the genus Aspergillus, Rhizopus, Trichoderma, Neurospora, Mucor, Penicillium, etc., such as yeasts of the genera Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, Hansenula, Pichia and the like. Suitable host systems are, for example, fungi such as Aspergilli, for example Aspergillus niger (ATCC 9142) or Aspergillus ficuum (NRLL 3135) or Trichoderma (eg Trichoderma reesei QM6a) and yeasts such as Saccharomyces, for example Saccharomyces cerevisiae or Pichia, for example Pichia pastoris or Hansenula, for example H polymorphic (DSMZ 70277). Such microorganisms may be obtained from recognized depositories, eg the American Type Culture Collection (ATCC), the Centraalbureau voor Schimmelcultures (CBS) or the German Collection for Microorganisms and Cell Cultures GmbH (DSMZ) or any other depositories. The expression cassette may contain, in the 5'-3 'direction of transcription, a transcriptional and translational initiation region of the polynucleotide of the invention and a transcription and termination region that is functional in vivo or in vitro. The termination region may be native to the transcription initiation region or may be native or of other origin with respect to the polynucleotide. The regulatory sequences may be located upstream (5 'non-coding sequences), within (intron) or downstream (3' non-coding sequences) of a coding sequence, and associated with transcription, RNA processing or stability and / or translation of the coding sequence affect coding sequence. Regulator sequences may include, without limitation, enhancers, promoters, repressor binding sites, translational leader sequences, introns, or polyadenylation signal sequences. They may include natural and synthetic sequences as well as sequences that are a combination of synthetic and natural sequences.

The vector used in the invention may also comprise suitable sequences for amplification of expression.

Examples of promoters which can be used in the present invention are promoters known to control expression in the eukaryotic cells. Any promoters capable of expression in filamentous fungi can be used. Examples are a promoter which is strongly induced by starch or cellulose, e.g. A promoter for glucoamylase or a-amylase from the genus Aspergillus or for cellulase (cellobiohydrolase) from the genus Trichoderma, a promoter for enzymes in the glycolytic pathway, such as phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GPD) , etc. The cellobiohydrolase I, the cellobiohydrolase II, the amylase, the glucoamylase, the xylanase or the enolase promoter is preferred.

In addition to using a particular promoter, other types of elements can affect the expression of transgenes. In particular, introns have been shown to have the potential to enhance transgene expression.

The expression cassette may include other elements, for example, those that can be regulated by endogenous or exogenous elements, such as zinc finger proteins, including naturally occurring zinc finger proteins or zinc protein chimeric proteins. The expression cassette used in the invention may further contain enhancer elements or upstream promoter elements. Vectors for use in the invention can be engineered to contain an enhancer element. The constructs of the invention thus comprise the gene of interest together with a 3 'DNA sequence which acts as a signal to terminate transcription and allow polyadenylation of the mRNA thus obtained. Any signal sequences that allow secretion from the chosen host organism can be used. A preferred signal sequence is the phospholipase signal sequence from Aspergillus fumigatus or signal sequences derived therefrom for secretion from filamentous fungi. A specific leader sequence can also be used since the DNA sequence between the transcription start site and the start of the coding sequence, ie the untranslated leader sequence, can influence gene expression. Preferred leader sequences include sequences that direct the optimal expression of the attached gene, ie, they include a preferred consensus leader sequence that enhances or maintains mRNA stability and prevents inappropriate translation initiation. The choice of such sequences is well known to one skilled in the art.

To enhance the ability to identify the transformants, a selectable or screenable marker gene can be incorporated into the expression cassette. Such marker genes are well known to one skilled in the art.

The expression cassette or a vector construct containing the expression cassette is introduced into a host cell. A variety of techniques are available and well known to those skilled in the art for introducing constructs into a host cell. The transformation of microbial cells can be carried out using polyethylene glycol, calcium chloride, viral infection, DEAE-dextran, phage infections, electroporation and other methods known in the art. The transformation of fungi can be performed according to Penttila et al., Gene 61: 155-164, 1987. The introduction of a recombinant vector into yeasts can be carried out by per se known methods including electroporation, use of spheroplasts, lithium acetate and the like. Once the expression cassette or DNA sequence of the present invention is obtained, it can be inserted into vectors according to methods known per se in order to overexpress the encoded polypeptide in suitable host systems. However, DNA sequences as such can also be used to transform suitable host systems of the invention to achieve overexpression of the encoded polypeptide.

Once a DNA sequence of the invention has been expressed in a suitable host cell in a suitable medium, the encoded phospholipase can be secreted either from the medium if the phospholipase is secreted into the medium or from the host organism if the phospholipase is intracellularly e.g. B. is present in the periplasmic space, concentrated and / or isolated according to known methods. Known methods of separating the insoluble components of the culture medium and the biomass followed by methods for concentrating the phospholipase can be used to prepare concentrated phospholipase solutions or as a preparation for drying the phospholipase. For example, filtration methods or centrifugation methods may be used to separate the insoluble components followed by ultrafiltration methods for concentration or cross-flow filtration methods. Drying may be by freeze and spray drying, granulation, extrusion or other methods. Known methods of protein purification can be used to isolate the phospholipases of the present invention. For example, various chromatographic or gel chromatographic methods can be used singly or in combination. Depending on the host cell used in a recombinant production process, the enzyme of the invention may or may not be covalently modified by glycosylation. In eukaryotic cells, glycosylation of the secreted proteins serves to modulate protein folding, conformational stability, thermal stability, and resistance to proteolysis. With regard to a specific application of the phospholipase, a glycosylated variant of the enzyme may be preferred over a non-glycosylated variant. The invention also relates to isolated or substantially purified nucleic acid or protein compositions. An isolated and purified polynucleotide / polypeptide or segment thereof refers to a polynucleotide or polypeptide or segment thereof which is isolated from its native environment and in purified form for further use. An isolated polynucleic acid segment or Polypeptide may be in purified form or may be in a non-native environment, such as in a transgenic host cell. For example, an isolated or purified polynucleotide segment or protein or biologically active portion thereof is substantially free of further cellular material or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemical compounds. Preferably, an isolated polynucleotide is free of sequences (preferably protein coding sequences) that naturally derive the nucleic acid (ie, sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived flank. For example, according to various embodiments, the isolated nucleic acid molecule may comprise less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb nucleotide sequences that naturally comprise the nucleic acid molecule in the genomic DNA of the cell flanking the nucleic acid is derived. A protein that is substantially free of cellular material includes protein or polypeptide compositions having less than about 70%, 50%, 30%, 20%, 10%, 5% (by dry weight) of contaminating protein. Preferably, when the protein or a biologically active fragment thereof of the invention is recombinantly produced, the culture medium comprises less than about 70%, 50%, 30%, 20%, 10%, or 5% (by dry weight) of the chemical precursors or not. proteinaceous chemical substances.

The invention also relates to phospholipase compositions containing the polypeptide of the invention. In general, phospholipase compositions are liquid or dry. Liquid compositions preferably contain the phospholipase enzyme in a purified or enriched form. However, adjuvants such as a stabilizer and / or glycerin, sorbitol or monopropylene glycol, additives such as salts, sugars, preservatives, pH adjusters and proteins may also be added. Typical liquid compositions are aqueous or oily slurries.

Dry compositions may be freeze-dried, spray-dried, granulated or extruded compositions which may contain only the enzyme. Dry compositions can be granules that can be easily blended with, for example, food or feed components, or more preferably form a component of a premix. The particle size of the enzyme granules is preferably that of the other components of the mixture compatible. This allows safe and convenient means to incorporate enzymes into, for example, processed foods, premixes or animal feed.

Dry compositions may also contain other additives, e.g. Salts, in particular phosphate salts and their anhydroforms, and stabilizers such as polyvinylpyrrolidone, etc., contain certain conditions, such as e.g. to regulate the pH in the application.

Similarly, a food additive according to this embodiment of the present invention may be combined with other food components to produce processed food products. Such other food components include one or more enzyme supplements, vitamins, minerals and trace elements. The combined dietary supplement thus obtained may then be mixed in an appropriate amount with other food components, such as cereals and vegetable proteins, to yield a processed food. The processing of these components into a processed foodstuff may be carried out using processing devices known per se. In a preferred embodiment, the phospholipase compositions of the invention additionally comprise an effective amount of one or more enzymes for food or feed or for use in precursors for the production of food or feed, or for use in the textile industry, preferably selected from alpha-galactosidases, beta-galactosidases, laccases, other phospholipases, phosphatases, endoglucanases, in particular endo-beta-1, 4-glucanases, endo-beta-1, 3 (4) -glucanases, endo-1, 2-beta-glucanases and endo-1 , 3-alpha-glucanases, cellulases, xylosidases, galactanases, in particular arabinogalactan-endo-1, 4-beta-galactosidases and arabinogalactan-endo-1,3-beta-galactosidases, pectin degrading enzymes, especially pectinases, pectin esterases, pectin lyases, Polygalacturonases, arabananases, rhamnogalacturonases,

Rhamnogalacturonanacetylesterases, rhamnogalacturonan-alpha-rhamnosidases, pectatlyases and alpha-galacturonidases, mannanases, beta-mannosidases, mannanacetylesterases, xylanacetylesterases, proteases, xylanases, arabinoxylanases, lipolytic enzymes such as lipases, digalactoside-diglyceride esterases and cutinases, and other enzymes such as laccases and transglutaminases. The phospholipases of the invention can be used for a variety of applications. Examples include applications in baking and animal nutrition and in the production of fuels from renewable energy sources, such as rapeseed, and in the processing of textile raw materials.

A preferred application is the use of the polypeptides of the invention having phospholipase activity in methods of degumming vegetable oil. In this case, for example, the edible oil to be degummed is treated with a polypeptide according to the invention, whereby the main part of the phospholipids is hydrolyzed, and then the aqueous phase containing the hydrolyzed phospholipids is separated from the oil. Such a process is particularly suitable for the purification of edible oils containing phospholipids, for example vegetable oils such as soybean oil, rapeseed oil and sunflower oil.

Prior to phospholipase treatment, the oil is preferably pretreated to remove mucilage, for example, by wet refining. Typically, the oil will contain 50 to 850 ppm of phosphorous phospholipid at the beginning of treatment with the phospholipase of the present invention. After treatment, the phosphorus value is typically between 2 and 10 ppm.

The phospholipase treatment is usually carried out so that the phospholipase is dispersed in an aqueous solution, preferably as droplets with an average diameter of <10 μm. The amount of water is preferably 0.5 to 5 wt .-% (w / w) based on the oil. Optionally, an emulsifier may be added. It can be stirred mechanically to maintain an emulsion. The treatment with phospholipase may be carried out at a pH in the range of about 3.5 to about 5.0. The process pH may range from about 3.5 to about 5, preferably 3.8 to 4.5, and most preferably 4.0 to 4.2 in order to maximize the performance of the enzyme. The pH can be adjusted, for example, by adding citric acid, a citrate buffer, phosphoric acid or hydrochloric acid. A suitable temperature is generally 30 ° -70 ° C, preferably 45 ° -65 ° C, and most preferably 55 ° -62 ° C. The reaction time is typically 1 to 12 hours, preferably 2 to 6 hours. A suitable enzyme dosage is usually 120 to 3000 units per kg of oil, preferably 250 to 2000 and most preferably 750 to 1500 units per kg of oil. The phospholipase treatment can be carried out in batches, for example in a tank with stirring, or it can be continuous, for example in a series of tank reactors with stirring. The phospholipase treatment is followed by the separation of an aqueous phase and an oil phase. The separation can be carried out by conventional means, for example centrifugation. The aqueous phase contains phospholipases and the enzyme can be reused to improve the economy of the process.

The treatment can be carried out using methods known per se.

The phospholipase according to the invention can also be advantageously used for the preparation of dough and bakery products, wherein an effective amount of a polypeptide according to the invention is incorporated in the dough. By adding a polypeptide of the invention having phospholipase activity, one or more properties of the dough or baked product obtained from the dough can be improved as compared to a dough or baked product without the addition of a polypeptide of the invention having phospholipase activity.

In dough making using the phospholipase of the invention, the phospholipase can be added to the dough itself, any ingredient from which the dough is made, and / or a mixture of dough ingredients from which the dough is made. A polypeptide with phospholipase activity according to the invention can thus be added as such at any stage of the dough preparation or can be added in one, two or more stages. An effective amount is meant herein to refer to an amount of phospholipase sufficient to produce a measurable effect on at least one property of interest in the dough and / or the baked product.

The term "improved property" is defined herein as any property of the dough and / or a product obtained from the dough, in particular a baked good, which is defined by the action of the phospholipase relative to the dough or product containing the phospholipase of the invention The improved property may include, for example: increased dough strength, increased dough elasticity, improved dough stability, reduced dough stickiness, improved extensibility of the dough, improved machinability of the dough, increased volume of the baked product, improved Crumb structure of the baked product, improved softness of the baked product, improved flavor of the baked product and / or delayed staling of the baked product. Methods for determining these properties are well known in the art.

A dough is defined here as a mixture of flour and other ingredients that is solid enough to be kneaded or rolled. The dough may be freshly frozen, pre-cooked or prebaked. As used herein, the term "baked product" refers to any product made from a dough that is either soft or crispy in character, and examples of baked products that can be made using a phospholipase of the invention include bread (especially white bread, wholemeal bread, or rye bread). typically French loaf or baguette, pasta, pita bread, tortillas, tacos, cakes, pancakes, biscuits and biscuits, cooked bread, double-baked bread and the like.

In the preparation of these baked goods, the polypeptide according to the invention having phospholipase activity and / or one or more further enzymes may be added in any formulations which are suitable for the particular use, for example in dry form, as a liquid or as a premix. Further, one or more other enzymes may also be added to the dough. These other enzymes can be of any origin and are derived, for example, from mammals and plants. Preferably, they are of microbial origin and more preferably are derived from bacteria or fungi.

According to a preferred embodiment, the further enzymes may include amylases such as α-amylase (suitable for producing sugars fermentable by yeast and delaying staling) or β-amylase, cyclodextrin glucanotransferase, peptidase, in particular an exopeptidase (suitable for enhancing aroma ), Transglutaminase, lipase (useful for modifying the lipids present in the dough or dough ingredients to soften the dough), phospholipase (useful for modifying the lipids present in the dough or dough ingredients to soften the dough) and improving gas retention in the dough), cellulase, hemicellulase, in particular a pentosanase such as xylanase (useful for the partial hydrolysis of pentosans, which improve the extensibility of the dough), proteases (useful for gluten softening, especially when using durum wheat flour), Protein disulphide isomerase (for example, a protein disulphide isomerase disclosed in WO95 / 00636), glycosyltransferase, peroxidase (useful for improving dough consistency, laccase or oxidase, for example aldose oxidase, glucose oxidase, pyranose oxidase, lipoxygenase or L-amino acid oxidase (useful for improving the consistency of the dough).

These optionally further added enzyme (s) can be added separately or together with the polypeptide according to the invention having phospholipase activity, optionally as constituents of baking or dough auxiliary. The invention also relates to the production of such doughs and the production of corresponding baked goods from these doughs.

The invention further relates to a premix, for example in the form of a flour composition, for the production of dough and / or baked goods from dough, this premix comprising polypeptides according to the invention having phospholipase activity.

The polypeptides with phospholipase activity according to the invention can also be used as an additive to animal feed. The addition of phospholipases to feed improves the efficiency of feed utilization in animals. This improves the growth of the animals fed with such feed. In this case, a phospholipase according to the invention can be added as such or as feed concentrate. Further, the phospholipase may also be added via transgenic plants to the animal feed wherein the phospholipase has been synthesized by heterologous gene expression. Methods for producing such transgenic plants are disclosed in EP0449376.

The polypeptides with phospholipase activity according to the invention can also be used in the process of scouring in the textile raw material processing of eg cotton fibers in order to facilitate the further processing of the fibers. The improvements achieved by the scouring effect on the behavior during staining as well as the further mechanical and enzymatic processing of the fiber as well as the fabric made from it. The gene for the phospholipase isolated from the microorganism Aspergillus fumigatus was in the plasmid pPL3949-Topo2.5 under the accession number DSM 22741 on 02/07/2009 at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Inhoffenstraße 7B, D-38124 Braunschweig deposited under the terms of the Budapest Treaty. The invention will be explained in more detail with reference to the accompanying figures. Show it:

FIG. 1: IEF gel of purified phospholipase from Aspergillus fumigatus.

Lane 1: Marker protein from the Isoelectric Focusing Calibration Kit, pH 2.5-6.5 Lane 2-3: The phospholipase band at pl ca. 4.1 is marked with an arrow. Figure 2: T-Optimum curve for the recombinant phospholipase expressed in Trichoderma reesei RH32664.

FIG. 3: pH optimum curve for the recombinant phospholipase expressed in Trichoderma reesei RH32664.

FIG. 4: Nucleotide sequence and amino acid sequence derived therefrom of the chromosomal phospholipase gene from Aspergillus fumigatus RH3949. The introns are in italics and the amino acid sequence in bold. (SEQ ID NO: 1)

FIG. 5: The nucleotide sequence of the chromosomal phospholipase gene from Aspergillus fumigatus RH3949 (SEQ ID NO: 1).

FIG. 6: The amino acid sequence of the phospholipase gene from Aspergillus fumigatus RH3949 (SEQ ID NO: 2)

FIG. 7: Restriction map of the vector pPL3949-Topo2.5

FIG. 8: Restriction map of the expression vector pAB500-PL3949

Reference Example 1 Determination of phospholipase activity

1 Phospholipase Unit (PLU) corresponds to the amount of enzyme which liberates 1 μιτιοΙ fatty acid per minute from phosphatidylcholine under standard conditions. reagents:

Substrate emulsion:

 1 g of Epikuron 200 (purified phosphatidylcholine from soy from Lucas Meyer, now available from Cargill), 100 ml of deionized water and 5 ml of 0.32 M CaCl 2 solution are homogenized with an Ultra Turrax for 2 min at 24,000 rpm.

 The substrate emulsion is stable at 4 ° -8 ° C for 3-4 days.

Other solutions:

 0.32 MgC solution, fresh 3.3 mM citric acid monohydrate solution, 10 mM KOH solution, 1% Triton X100 (from Fluka) solution in demineralized water

Enzyme solution:

The enzyme preparations are in deionized water The enzyme concentration in the mixture must not exceed 2.5 U g ^"1 lie.

Execution:

home values

 10 ml substrate emulsion

 10 ml of 1% Triton X100 solution

 5 ml of 3.3 mM citric acid monohydrate solution

were pipetted into a 25 ml Weithalserlenmeyer flask and heated to 40 ° C for 10 min. The pH value is about 3.3-3.5.

 After the addition of 0.1 ml of enzyme solution, the analysis batch was incubated at 40 ° C. for 10 min. After the incubation period is titrated with 10 mM KOH to pH 10.0, the first 5 ml of KOH are added quickly (duration: about 1 min). The consumption of KOH is registered.

blanks

 The enzyme stock solution is heated for 15 min at 95 ° C and thus deactivated. After cooling to room temperature, the further treatment is carried out as in the main values.

 An incubation of the blank values is not necessary. evaluation

 AY KOH * CKOH * \ 000

 PLU / g

 At * Cs * v

VKOH (ml) Consumption difference between blind and main value

CKOH (mol ¹ ) concentration of KOH solution

At (min) incubation period cs (g ml ^"1 ) Concentration of the sample

v (ml) volume used

Reference Example 2

 Phospholipase rapid test at pH 3.5

Reagents: Substrate emulsion:

 1 g Epikuron-200 are mixed with 100 g Milli Q water and 5 ml 0.32 M calcium chloride solution and homogenized with the Ultra-Turrax (about 1 -2 min at about 24000 rpm).

 For the analysis approach, 10 ml of this substrate emulsion with 10 ml of 1% Triton X-100 solution and 5 ml

 3.3 mM citric acid monohydrate solution.

Free fatty acids, semi-micro test (Boehringer)

 (The half-micro test contains the reagents for the reaction mixture A, reaction mixture B and the N-ethylmaleimide)

Execution:

5 μΙ diluted enzyme solution are placed in a microtiter plate and mixed with 0.1 ml of substrate emulsion.

 The substrate / enzyme mixtures are incubated for 10 min at 40 ° C in a water bath.

 Subsequently, 5 μΙ of the batch in a second microtiter plate to 100 μΙ

Reaction mixture A pipetted and incubated for 5 min at 40 ° C in a water bath.

 After the incubation period 5 μΙ of a mixture in the ratio 1: 1 of reaction mixture B and N-ethylmaleimide solution are added and again for 5 min at

40 ° C incubated.

 Phospholipase activity is indicated by a red coloration of the reaction mixture.

Reference Example 3

Determination of the content of free fatty acids

reagents:

 Ethanol 96%, toluene,

Ethanol and toluene are mixed in the ratio 1: (v / v). 0.1 N ethanolic KOH

 1% phenolphthalein solution in ethanol

Execution:

Approximately 3 g of anhydrous oil are weighed exactly to 4 decimal places in an Erlenmeyer flask, dissolved in 20 ml of ethanol-toluene mixture, admixed with 2 to 3 drops of phenolphthalein and titrated with 0.1 N KOH solution to a permanent red color.

Evaluation:

The acid number is a measure of the free fatty acid content. The acid number denotes the amount of potassium hydroxide in g necessary to neutralize free fatty acids contained in 1 kg of oil. The acid number (SZ) is calculated according to the following equation: a * N ^* 56.1

 SZ =

 e

a consumption of KOH solution in ml

 N normalcy of KOH

 E fat scale in g

 56, 1 molar mass of KOH in g / mol

From the acid value, the percentage of free fatty acids (FFA) can be calculated:

FFA (%) = SZ ^* 282 ^* 100

 56.1 000

282 Molar mass of oleic acid Reference Example 4 Lipase detection

The qualitative lipase detection on olive oil agar is carried out analogously according to the method of Kouker and Jaeger (Applied Environ. Microbiol., 59: 21 1 -213 (1987)). To detect lipase activity, agar plates prepared from Tributyrinagar (Fluka 91015) with 1% olive oil are used. The pH value is 5.5.

The lipase activity is carried out photometrically with emulsified p-nitrophenyl palmitate (Sigma N2752) as substrate in 0.5 M citrate / phosphate buffer, pH 5.1 analogously to Winkler and Stuckmann (1979) (J. Bac, 138: 663-670 (1979) ).

example 1

 Obtaining phospholipase from Aspergillus fumigatus strain RH3949

Aspergillus fumigatus was grown in 200 ml shake flasks filled with 50 ml of medium at 28 ° C, 200 rpm, for 5 d. The medium consisted of 0.5% Epicuron 200 (Lucas Meyer), 0.5% cornstarch, 0.2% NH ₄ NO ₃ , 100 mM KH ₂ PO ₄ and 0.1% Triton X100. The pH was adjusted to pH 6 before sterilization. The medium was inoculated with a spore suspension. After 5 days, the culture supernatant was separated from the mycelium by filtration and the phospholipase activity in the fluid was measured.

Example 2

 Purification of phospholipase from Aspergillus fumigatus strain RH3949 Step 1: Anion exchanger, Macro Prep Q

For the purification of the phospholipase, first a separation of the proteins on the anion exchanger Macro Prep Q was carried out.

 For this purpose, the concentrated culture supernatant from the cultures of Example 1 was diluted with demineralized water until the protein solution had the same conductivity as the conductivity of the buffer A. Subsequently, the protein sample was adjusted to pH 7 with 1 M NaOH and loaded onto the column equilibrated with buffer A. After washing the column with buffer A, the phospholipase was eluted with a linearly increasing NaCl gradient of 0-1 M. The fractions with phospholipase activity were pooled and further purified.

Buffer A: 5 mM CaCl ₂ + 20 mM Tris-HCl, pH 7.0

Buffer B: 5 mM CaCl ₂ + 20 mM Tris-HCl, pH 7.0 + 1 M NaCl Step 2: HIC, Phenyl Sepharose 6 Fast Flow Low Substitution

The protein sample with phospholipase activity from step 1 was mixed 1: 1 with 3.4 M ammonium sulfate solution and adjusted to pH 7.0 with 1 M NaOH solution. After applying the sample to the phenyl Sepharose column also equilibrated with buffer A, the phospholipase was eluted with a decreasing ammonium sulfate gradient.

Buffer A: 5 mM CaCl ₂ + 20 mM Tris-HCl, pH 7.0 + 1.7 M ammonium sulfate

Buffer B: 5 mM CaCl ₂ + 20 mM Tris-HCl, pH 7.0

Step 3: Gel filtration, Superose 12 HR 10/30

The last purification step was a separation of the proteins on a gel filtration column. For this purpose, the phospholipase sample from step 2 in the dialysis tube (Naturin protein Saitling) was dialyzed against demineralized water for 1 .5 h and then lyophilized. The lyophilizate was taken up in 500 μΙ of demineralized water. In two runs, 250 μΙ each were applied to the column and eluted with the buffer A.

Buffer A: 5 mM CaCl ₂ + 20 mM Tris-HCl, pH 7.0

The purified phospholipase was applied to an IEF gel. The result is shown in FIG.

For identification, the bands were excised and checked for phospholipase activity according to the analysis method described.

Example 3 N-terminal protein sequencing

After the last purification step on the Superose, the purified protein was separated on a native gel. The protein bands with phospholipase activity were excised and reapplied to an SDS gel to determine the molecular weight. For N-terminal amino acid determination, the protein bands from the native gel were transferred to a PVDF membrane (Fluotrans Transfer Membrane, Pall) and after Coomassie staining the N-terminal amino acid sequences were determined in an amino acid sequencer (Applied Biosystems Model 470A). They are: ¹ DVSAS VLQKL SLFAQ Y ¹⁶ (SEQ ID NO: 3)

The sequence comparison shows that the N-terminal amino acid sequence of the phospholipase gene has a high sequence relationship with the extracellular lipase gene from the Aspergillus fumigatus strain Af293 (GenBank EAL86100).

Example 4

Cloning of the chromosomal phospholipase gene from Aspergillus fumigatus strain RH3949 by polymerase chain reaction (PCR)

From the data of the chromosomal DNA sequence of the Aspergillus fumigatuspase, various oligoprimers for the amplification of the phospholipase DNA were derived. The chromosomal DNA preparation was prepared according to a modified protocol of Hynes, M.J et al., (1983) Mol. Cell. Biol. 3, 1430-1439. The amplification of a phospholipase gene was carried out by the PCR method. The PCR products were cloned into pCR2.1 -TOPO plasmid and sequenced. It shows that the primer pair N2-3948 and 3949-Apal leads to the gene with the phospholipase DNA according to the invention.

N2-3948 5 ^' - AGAGTCTGCC TATATTCTCT CTGAAAGG -3 ^' (SEQ ID NO 4) 3949-Apal 5 ^' -TATGACAATTTCCGTGATTACTG-3 ^' (SEQ ID NO: 5)

The reaction mixture of 100 .mu.l contained: 10 .mu.l 10 x buffer (200 mM Tris / HCl, pH 8.4, 500 mM KCl), 3 .mu.l 50mM MgCl ₂ , 2 .mu.l 10 mM dNTP, each 50 pmol Oligoprimer (N2 3948 and 3949 Apal), approximately 10 ng of chromosomal DNA, 5U Taq DNA polymerase (Invitrogen). The batch was for denaturation at 95 ° C / 5 min, 45 cycles (95 ° C / 1 min, 45 ° C / 1 min, 72 ° C / 1 min) and then the extension at 72 ° C / 10 min performed ,

The PCR products were purified on Qiaquick column and cloned into pCR2.1 -TOPO plasmid. A transformant after sequencing contained the phospholipase DNA sequence of the invention (Figure 5, SEQ ID NO: 1) and was designated pPL3949 topo2.5 (Figure 7).

The open reading frame encoding the phospholipase comprises 1052 nucleotides containing 299 amino acids. The phospholipase gene contains 3 introns. The deduced N-terminal amino acid sequence is consistent with the peptide sequences determined from protein sequencing (Example 3, SEQ ID NO: 3).

The deduced molecular weight of about 28.6 kDa corresponds to the approximately 29 kDa determined by SDS-PAGE (Example 8).

The determination of the signal sequence was carried out with a computer program (PSORT) by Nakai and Kanehisa (1992, Genomics 14, 897-91 1). Subsequently, the phospholipase gene has a signal sequence of 21 amino acids and a propeptide of 8 amino acids.

Example 5

Construction of expression vector pAB500-PL3949

In the expression vector pAB500-PL3949, the phospholipase gene is under the control of the T. reesei cbhl promoter and cbhl terminator.

 For the construction of the plasmid pAB500-PL3949, the phospholipase-encoding gene was amplified from the plasmid pPL3949.ToPO2.5 by PCR.

 The PCR product was hydrolyzed with the enzymes Avrll / Pacl and then inserted into the Spei and Pacl cleavage sites after the T. reesei cbhl promoter in the plasmid pAB500. The resulting plasmid has the name pAB500-PL3949. The construction of the plasmid pAB500 was carried out by the following steps:

 The plasmid pAB487 was prepared from the plasmid pALK487 (WO94 / 281 17) by inserting further interfaces (Spei and Pacl in the SacII site between the cbhl promoter and cbhl terminator). The Spel-Pacl interfaces are used for the direct cloning of the phospholipase gene.

The amdS gene including its promoter and its terminator was amplified from the plasmid p3SR2 (GenBank 16371) by PCR. The PCR product was cut with Asel and Nrul and inserted into the Asel / Stul cleavage site of pAB487 to give the plasmid pAB500. Example 6

 Transformation of T. reesei

The techniques used to transform and handle T. reesei were according to Penttilä et al. (1987, Gene 61: 155-164).

 T. reesei RH32439 was transformed with the linearized expression cassette isolated from plasmid pAB500-PL3949.

 The transformants were selected and purified by single spore isolation. Of all the transformants, those with the highest secretion efficiency were selected and further used in Example 7 for the production of enzyme material.

Example 7

 Preparation of Enzyme Solutions by Fermentation in Shake Flasks Transformants carrying the expression cassette of Example 6 were cultured in shake flasks on cellulase-inducing medium. The culture filtrates obtained after 6 days of culture were used for the characterization of the enzyme (Example 8) and for the analysis of the oil degumming (Example 9). Example 8

 Characterization of the recombinant phospholipases

The molecular weight determination by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of about 29 kDa for the phospholipase according to the invention.

N-terminal sequencing of the recombinant phospholipase was performed by Chromatec (Germany). The amino acid sequence obtained by the sequencing is in agreement with the N-terminal sequence of the phospholipase (Example 3).

The identification of the recombinant protein with MALDI-MS was carried out by Protagen (Germany). The result confirms the correctness of the protein sequence of the phospholipase according to the invention.

The temperature dependence of the enzyme activity was determined by the determination method as described above at various temperatures. The temperature optimum of the phospholipase is 50 ° C (see Figure 2). The optimum pH of the enzyme activity was determined by the determination method as described above at various pH values. The pH in the reaction mixture was adjusted with the aid of citric acid. The enzyme is active in a wide pH range of pH 3-5 (see Figure 3).

The lipase activity was determined by the method of determination described above. The A. fumigatus phospholipase according to the invention has a very low lipase activity. The ratio of phospholipase to lipase activity was determined to be 7,480: 1 (± 10% experimental variation).

Example 9

 Degumming of oil with enzyme from culture supernatants of recombinant Trichoderma reseei strains with the gene from A. fumigatus RH 3949

In a 400ml beaker was added 200 grams of edible oil (soybean oil 1 with 163.6 ppm phosphorus content, rapeseed oil with 161, 6 ppm phosphorus content, soybean oil 2 with 592.8 ppm phosphorus content and soya oil 3 with 81.1 ppm phosphorus content) with 0.42 ml of 46% Citric acid solution and water. The total water content of 2% should not be exceeded. The pH of the reaction mixture was adjusted to pH 4.0 by adding 7% NaOH solution (0.6 ml). After addition of the enzyme solution according to the invention (50 U), the reaction mixture was mixed with the aid of an Ultra Turrax for 2 min. At 24000 rpm. The mixture was then transferred to a 3-neck round bottom flask and incubated with gentle stirring (200 rpm) at 55 ° C and 60 ° C, respectively.

The sampling of 20 ml took place every 120 min after addition of the enzyme solution. The samples were centrifuged for 5 min at 4300 x g and the phospholipid content, expressed in ppm of phosphorus, was determined photochemically at 830 nm in the oil after ashing at 850 ° C with the addition of magnesium oxide as a phosphomolybdate complex.

The recombinant Trichoderma reesei strain containing the plasmid pAB500-PL3949 according to the invention is designated RH32664.

The results of degumming edible oil at 55 ° C. or 60 ° C. with recombinant strain-produced phospholipase from Aspergillus fumigatus RH 3949 are shown in Table 2. The results show a significant degumming effect by the enzyme according to the invention in comparison with the water degumming with citric acid. After 4 hours, the residual phosphorus content is less than 10 ppm. Tab .: 2: Degumming of soybean oil and rapeseed oil with phospholipase enzymes according to the invention (250 U per kg / edible oil) from culture supernatants of recombinant Trichoderma reesei strain with 2% water content at pH 4.0

Example 10

 Free fatty acid content in the oil degumming process

The determination of the free fatty acid content in the oil degumming process was carried out as described in Reference Example 3. The edible oil was mixed with phospholipase as in Example 9 and incubated at 57 ° C for 6 hours. The enzymatic hydrolysis of phospholipids was caused by phospholipase, which split off the fatty acid. For comparison, the same analysis was performed with pure edible oils as a blank.

Compared to the blank value (BW), the free fatty acid (FFA) content in the samples increases only slightly and the difference (A _F FA) is 0.18% after the treatment of edible oil with phospholipase at a temperature of 57 ° C (Table 3).

Tab. 3: Free fatty acid content after treatment of the oil with phospholipase.

Claims

claims

1 . A DNA sequence encoding a polypeptide having phospholipase activity substantially without lipase activity, characterized in that the DNA sequence is selected from

 a) DNA sequences comprising a nucleotide sequence according to SEQ ID NO: 1,

 b) DNA sequences which comprise the coding sequence according to SEQ ID NO: 1,

 c) DNA sequences which encode the protein sequence according to SEQ ID NO: 2, d) DNA sequences which are encoded by the plasmid pPL3940-Topo2.5 with the restriction map according to FIG. 7 and deposited under the accession number DSM 22741,

 e) DNA sequences which hybridize under stringent conditions with one of the DNA sequences according to a), b), c) or d),

 f) DNA sequences which are related due to the degeneracy of the genetic code with the DNA sequences according to a), b), c), d) or e), and

 g) complementary strands to the sequences according to a) to f),

 wherein the DNA sequence is preferably derived from Aspergillus and more preferably Aspergillus fumigatus.

2. A nucleic acid sequence comprising an analogue of any of the sequences according to claim 1, characterized in that the sequence encodes a polypeptide having phospholipase activity substantially without lipase activity, and

 a) has at least 82% identity to one of the sequences according to a), d), e), f) and g), or

 b) has at least 88% identity to one of the sequences according to b), c), e), f) and g), or

 c) hybridizing with one of these sequences under stringent conditions, or d) is an allelic variant of one of these sequences, or

e) is a complementary strand to the sequences according to a) to d), wherein the sequence is obtainable by substitution, deletion, insertion, addition or mutation of one or more nucleic acids of the DNA sequence according to SEQ ID NO: 1.

3. An expression construct comprising a sequence according to any one of claims 1 to 2 operably linked to one or more sequences for directing expression of the polypeptide having phospholipase activity in a suitable host cell, the sequence for controlling the expression of the polypeptide preferably being a promoter, selected from the glucoamylase or a-amylase promoter of the genus Aspergillus, the cellulase (cellobiohydrolase) promoter of the genus Trichoderma, a promoter for an enzyme in the glycolytic pathway such as phosphoglycerate kinase or glyceraldehyde-3-phosphate dehydrogenase, the xylanase promoter or the enolase Promoter is and optionally further comprising a secretory leader sequence.

4. Recombinant host cell, characterized in that it has been transformed with an expression construct according to claim 3.

5. Recombinant host cell according to claim 4, characterized in that it is derived from a fungal cell of the genus Aspergillus, Rhizopus, Trichoderma, Neurospora,

Mucor or Penicillium or a yeast cell of the genus Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, Hansenula or Pichia.

6. Plasmid pPL3949-Topo2.5 with the restriction map according to FIG. 7 and deposited under the accession number DSM 22741.

7. Polypeptide with phospholipase activity substantially without lipase activity selected from

 a) a polypeptide which is encoded by the coding part of a DNA sequence according to any one of claims 1 to 2,

b) a polypeptide having the sequence according to SEQ ID NO: 2 or a sequence derived therefrom obtainable by substitution, addition, deletion of one or more amino acids thereof, c) a polypeptide having a sequence which has at least 83% identity to the amino acids 1 to 299 of SEQ ID NO: 2,

 d) a polypeptide encoded by a nucleic acid sequence which hybridizes under stringent conditions

 (i) nucleotides 55 to 1106 of SEQ ID NO: 1,

 (ii) the cDNA sequence contained in nucleotides 55 to 1106 of SEQ ID NO: 1,

 (iii) a partial sequence of (i) or (ii) of at least 100 nucleotides, or

 (iv) a complementary strand of (i), (ii) or (iii),

 e) a variant of the polypeptide of SEQ ID NO: 2 comprising a substitution, deletion and / or insertion of one or more amino acids,

 f) allelic variants to the amino acid sequences a) to e).

8. Polypeptide with phospholipase activity, characterized in that it

- Has a molecular weight in the range of 28 to 30 kDa

 - be able to hydrolyze at least one of the two fatty acids from lecithin,

 has substantially no lipase activity,

 - has a high temperature resistance and

 - Is isolated from an organism of the genus Aspergillus.

9. Polypeptide according to claim 8, characterized in that it is isolatable from Aspergillus fumigatus.

10. Polypeptide according to claim 9, characterized in that it is immunologically reactive with an antibody directed against the sequence SEQ ID NO: 2.

1 1. Polypeptide according to one of claims 8 to 10, characterized in that it comprises the sequence according to SEQ ID NO: 2.

12. A phospholipase composition, characterized in that it comprises a polypeptide according to any one of claims 7 to 1 1 together with excipients and optionally further comprising one or more enzymes for food or feed.

13. Use of a polypeptide according to any one of claims 7 to 1 1 or a phospholipase composition according to claim 12 for the degumming of vegetable oil, for the production of dough and / or baked goods, for the production of dairy products, as an additive to animal feed or for the treatment of textile raw materials.

14. A process for the production of a polypeptide with phospholipase activity according to any one of claims 7 to 1 1, characterized in that a host cell according to any one of claims 4 or 5 is cultivated under conditions that promote the expression of the polypeptide, and then the polypeptide wins.

15. A method for degumming of vegetable oil, characterized in that

 a) optionally pretreating the vegetable oil to be treated,

 b) adding to the optionally pretreated oil an aqueous solution containing a polypeptide according to any one of claims 7 to 11 or a phospholipase composition according to claim 12,

 c) the reaction mixture according to b) heated to a temperature between 30 ° and 70 ° C for 1 to 12 h, and

 d) separating the aqueous and oily phases from each other.